The plant Artemisia annua (family: Asteraceae) produces a sesquiterpenoid lactone endoperoxide named artemisinin which is a promising antimalarial drug effective against Plasmodium falciparum, Plasmodium vivax at nanomolar concentration. Artemisinins are active against Schistosoma mansoni and S. japonicum in-vitro and in-vivo in experiments in animals. These schistosomes, like malarial parasites, degrade haemoglobin and produce hemozoin. These compounds are also active against Leishmania major, Toxoplasma gondii and Pnenmocystic carinii in-vitro and against P. carinii in-vivo. Artemisinins have immunosuppressive activity and also potential anticancer activity. For these activities, the doses of artemisinin required are substantially higher than the dose for antimalarial activities. According to Meshnick et at., (1996) (Microbiological Reviews 6:301-315) the antimalarial endoperoxides including artemisinin, dihydroartemisinin and arteethers, are not likely to be useful for other therapeutic purposes except against malarial parasites.
Fluoroquinolones, such as ciprofloxacin and ofloxacin are currently used against multi-drug resistant tuberculosis as alternatives to the first line agents such as isoniazid, streptomycin and rifampicin (Drlica et al., 1995; In. W. Rom and S. Garay (ed), Tuberculosis. Little, Brown & Co., Boston). However, M. tuberculosis frequently tends to acquire resistance to the quinolones, limiting their clinical usefulness (Tsukamura et al., 1985, Am, Rev. Respir. Dis. 131:352-56). Therefore, newer drugs which are effective against quinolone resistant pathogenic bacteria need to be evolved. It is also worthwhile to note that mycobacteria and E. coli respond to quinolone treatment in a similar fashion, justifying the extrapolation of the concepts developed with E. coli to mycobacteria. Both these bacteria develop resistance to quinolone drugs by virtue of point mutations in a short stretch of nucleotides known as the quinolone determining region within the gyrA gene encoding DNA gyrase subunit A (Cambau et al., 1994, Journal of Infactious Diseases 170: 479-83). Mycobacterium tuberculosis infects ten million people and kills three million each year making it the greatest cause of mortality by a single infectious agent worldwide. Quinolones have been shown to inhibit DNA gyrase activity such supercoiling and in turn DNA replication . Several investigators have shown that point mutations in gyrA gene occurring in very small region between 0-400 nucleotides of the gene are responsible for quinolone resistance acquired by different strains of E. coli selected either in-vitro or in vivo. The similarity of action of fluoroquinolones in Mycobacterium sp. and Haemophilus influenzae and the subsequent resistance mechanisms that have developed due to mutations in gyrA gene are related to E. coli. So the mechanism of quinolone resistance are similar between E. coli, Mycobacteria and H. influenzae (Georgiou et al.,1996, Antimicrobial Agents and Chemotherapy 40: 1741-1744 Cambau et al., 1994, The Journal of Infectious Diseases 170: 479-483).
During the past decade, bacteria that cause human diseases have been reported to be fast developing genetic resistance to many of the antibiotics commonly used for treatment (Witte 1998, Science 279: 996-998). All of the pathogens usually reported in hospitals including mycobacteria, pneumococci and enterobacteriaceae are known to develop drug resistance. Although, the emergence of drug resistance is a serious health concern, the appearance of multi-drug resistant strains of Mycobacterium tuberculosis (MDR-TB) is more disturbing since few drugs are effective against tuberculosis (Bloom et al.,1992, Science 257: 1055-1064; Heym et al., 1994, Lancet 344: 293-298). Such MDR-TB has resulted in fatal outbreaks in many countries, including the United States (Snider et al., 1992, New England Journal of Medicine 326: 703). Strains of MDR-TB which are resistant to as many as seven drugs have already been reported (Frieden et al., 1993, New England Journal of Medicine 328: 521). Development of bacterial resistance to drugs costs not only money but also human lives. Resistant infections are associated with increased morbidity, prolonged periods during which individuals are infectious and greater opportunity for the spread of infections to other individuals. The available antibiotics at present are usually of fungal origin against which a wide spectrum of drug resistance genes have already evolved.
Because of the omnipresence of the bacterial diversity in the biosphere, plants have always been exposed to bacterial infections. At the same time, plants exhibit natural resistance to bacterial attack and therefore possess compounds effective against pathogenic bacteria which have developed resistance to various drugs. Such plant compounds are classified as fourth generation antibiotics, useful in treating infectious diseases.
As mentioned earlier, artemisinin is effective against malaria. Although artemisinin rapidly suppresses the activity of parasites like plasmodium vivax and P. falciparum, problems with high rate of recrudescence (&gt;10% recrudescence infections), short half life persist. Hence, there is a need to develop new drugs against quinolone resistant pathogenic bacteria. It is a known fact that clinically used antibacterial broad spectrum compounds such as quinolones which exhibit DNA gyrase activity of mycobacterium sp. (causing tuberculosis) and Haemophilus sp. (causing tuberculosis) and Haemophilus influenzae are gradually becoming ineffective due to the occurrence of mutatious in gyrase genes and their natural selection under continuous use of such drug.
The Applicants, during their research screened a number of semisynthetic artemisinin related compounds to check the anti-bacterial and anti-fungal activity. The Applicants studied the nature of .alpha. and .beta. arteether, synthesised from dihydroartemisinin by etherification with ethanol. These compounds were developed as antimalarial drugs by Central Drug Research Institute (CDRI), Lucknow, India and Central Institute of Medicinal & Aromatic Plants (CIMAP), Lucknow, India, after phase II clinical trial. The absolute stereochemistry of arteethers at C-12 were also determined and it was found to be 2-3 times more potent than artemisinin. The Applicants during their research, came across .alpha.-arteether and .beta.-arteether out of which .alpha.-arteether was found to inhibit the growth of E-coli strains defective in DNA-gyrase enzyme. The applicants observed that DNA gyrase mutants were sensitive to .alpha.-arteether whereas the wild type of E-coli having intact DNA gyrase genes were not sensitive to said .alpha.-arteethers. In fact, the isomer of this compound, .beta.-arteether, does not exhibit this activity.
As such, the Applicants found a novel property of .alpha.-arteether as being effective against the gyr mutant strains of E. coli but ineffective against wild type strains. This will help to generate next generation antibiotics to kill the infectious bacteria which have already developed resistance to quinolones and fluoroquinolones. Hence, the Applicants also tested the efficacy of the compounds against fungi and found that it inhibits fungal growth due to its antifungal activity. The uses of artemisinin endoperoxide as an antimalarial drug and the potential use of the compound as antiprotozoan is well cited in the literature. However, the novel use of .alpha.arteether, a semisynthetic endoperoxide as a drug for bacterial infections caused by quinolone resistant bacteria and its use as an antifungal agent is novel and has not been reported earlier.
It is an object of the present invention to use .alpha.-arteether as a drug for gyr mutant bacteria which are resistant to quinolone drugs, and can be used as therapeutic agents for treating drug resistant bacterial infections.
It is yet another object of the invention to identify the antifungal function of an agent, the novel use of which was not known previously.
In accordance with the above objects, the present invention provides a method for treating bacterial or fungal infections in animals using .alpha.-arteether.